Human tissue-type plasminogen activator (t-PA) can dissolve local thrombi in blood vessels and recanalize a blocked vessel by activating plasminogen. At present, Genentech (USA) is the only entity that can produce recombinant human t-PA(rt-PA) at a large scale by means of genetic engineering technology and has developed successfully a rt-PA medicine. The first generation of rt-PA made in this company has become the treatment of choice for thrombotic diseases, but it has some shortcomings First, its price is rather high at $1375 per vial on the international market. Second, its function has some weak points: 1)short half-life, just 3 to 5 minutes; 2)low specific affinity with fibrin; 3) activity and efficacy should be further improved. A much higher dose should be given because of the short half-life. Low specificity and activity give rise to two results. On one hand, there is still 10-20% patient's thrombi that cannot be completely dissolved by rt-PA. On the other hand, it results in fibrinolysis over all the body and affects the normal blood clotting mechanism so as to cause severe complications such as bleeding, especially in the skull and digestive tract.
For the above reasons, some other companies have developed the second generation of rt-PA since the rt-PA was used in the clinical. They modified the rt-PA gene in order to eliminate or improve upon the above weak points. TNK-TPA is a kind of mutated form of rt-PA which is universally researched. For example, Keyt BA, Paoni NF, Refino rt CJ, et al proclaimed “a Faster-acting And More Potent Form of Tissue Plasminogen Activator” ((Proc Natl Acad Sci USA) 1997 Apr. 26; 91(9): 3670-4). It was constructed on the basis of three sites of the t-PA gene by site-directed mutation. Two sites are located in the Kringle function domain, T103N and Q117N; one site is located in the protease domain, “KHRR296-299AAAA” (K296A, H297A, R298A, R299A). T103N adds a glycosylation site which increases specificity of complexing of TNK-tPA and fibrin and decreases its cleanup rate; Q117N destroys combination of K1 region and mannose and the action domain of Kupffer cells in liver, and prolongs half-life; KHRR296-299AAAA destroys the functional region of PAI-1 and prolongs its half-life, too. As for the production of TNK-TPA, it is universal to adopt an ordinary eukaryotic gene expression vector, using CHO cells as the host cell. TNK-TPA integrates randomly into the expression system of the host cell genome. For example, Wu Benchuan, et al, announced the above expression system and preparation method of expressed protein in the article “Purification and identification of recombinant tissue-type plasminogen activator” published in <Development of Biochemistry and Biophysics> (1997, 24 (1):71-75).